Enterotoxigenic Escherichia coli (ETEC) is the major source of E. coli mediated 30 diarrhoea in humans and livestock. ETEC infections cause more than 280 million annual episodes of diarrhoea resulting in mortality numbers exceeding 300,000 deaths of children under the age of five years.
The significant negative health- and socio-economic impact of ETEC infection manifests itself mainly in the third world nations with poor sanitation and inadequate supplies of clean water. ETEC is a diverse group of pathogens defined by their ability to colonize the small intestine and secrete heat-labile and/or heat stable enterotoxins. The complex pathogenicity is further attributed to the presence of additional bacterial virulence genes on mobile genetic elements such as plasmids and chromosomal pathogenicity islands.
Much attention has been devoted to the understanding of how ETEC and other mucosa-associated pathogens interact with host tissue during infection. Recent work has revealed that bacterial protein glycosylation plays an important role in mediating adhesion, colonization and invasion of host tissue.
Up until now, the known protein glycosylation repertoire of E. coli was limited to just four proteins, all of which are surface-exposed adhesins with functions in bacterial pathogenesis. The prototypical ETEC strain H10407 encodes two known glycoproteins, TibA and EtpA.
While the intimate coupling between protein glycosylation and bacterial pathophysiology has become apparent, the discovery of novel glycoproteins implicated in virulence is only advancing slowly. This gap of knowledge is linked to the inherent challenges associated with glycoproteomics. The analytical tools developed for enrichment of eukaryotic 0- and N-linked glycopeptides rely on a limited set of defined physiochemical properties, e.g. glycan hydrophilicity or specific lectin recognition, which are relatively rare in bacteria.
Discovery and characterization of glycoproteins is further complicated by heterogeneous glycosylation, low abundance and poor ionization of peptides modified with carbohydrates compared to the non-modified counterpart.
Mapping of O-linked glycan moieties has proven to be a particularly challenging 35 task owing to the diverse nature of carbohydrate structures available for protein modification in bacteria. Although methods such as periodic acid/hydrazide glycan labelling and metabolic oligosaccharide engineering (MOE) have identified glycoproteins in a range of bacteria, these techniques present limitations in the form of low specificity for glycosylated proteins and dependence on sugar uptake and integration into bacterial glycoproteins, respectively.
Although they are poorly understood, bacterial glycoproteins potentially constitute an important reservoir of novel therapeutic targets, which could be used against bacterial pathogens.
Thus, there is a great need for understanding the glycosylation patterns of proteins originating from bacteria such as ETEC, and revealing the effect of the glycosylations on for example immunogenicity.